1. Field of the Invention
This invention is in the field of medicinal chemistry. In particular, the invention relates to methods for producing gossypol acetic acid co-crystals and (−)-gossypol acetic acid co-crystals. The invention also relates to pharmaceutical compositions comprising gossypol acetic acid co-crystals or (−)-gossypol acetic acid co-crystals and the use of gossypol acetic acid co-crystals and (−)-gossypol acetic acid co-crystals for inducing apoptosis in cells and for sensitizing cells to the induction of apoptotic cell death.
2. Related Art
The aggressive cancer cell phenotype is the result of a variety of genetic and epigenetic alterations leading to deregulation of intracellular signaling pathways (Ponder, Nature 411:336 (2001)). The commonality for all cancer cells, however, is their failure to execute an apoptotic program, and lack of appropriate apoptosis due to defects in the normal apoptosis machinery is a hallmark of cancer (Lowe et al., Carcinogenesis 21:485 (2000)). Most of the current cancer therapies, including chemotherapeutic agents, radiation, and immunotherapy, work by indirectly inducing apoptosis in cancer cells. The inability of cancer cells to execute an apoptotic program due to defects in the normal apoptotic machinery is thus often associated with an increase in resistance to chemotherapy, radiation, or immunotherapy-induced apoptosis. Primary or acquired resistance of human cancer of different origins to current treatment protocols due to apoptosis defects is a major problem in current cancer therapy (Lowe et al., Carcinogenesis 21:485 (2000); Nicholson, Nature 407:810 (2000)). Accordingly, current and future efforts towards designing and developing new molecular target-specific anticancer therapies to improve survival and quality of life of cancer patients must include strategies that specifically target cancer cell resistance to apoptosis. In this regard, targeting crucial negative regulators that play a central role in directly inhibiting apoptosis in cancer cells represents a highly promising therapeutic strategy for new anticancer drug design.
Two classes of central negative regulators of apoptosis have been identified. The first class of regulators is the inhibitor of apoptosis proteins (IAPs) (Deveraux et al., Genes Dev. 13:239 (1999); Salvesen et al., Nat. Rev. Mol. Cell. Biol. 3:401 (2002)). IAPs potently suppress apoptosis induced by a large variety of apoptotic stimuli, including chemotherapeutic agents, radiation, and immunotherapy in cancer cells.
The second class of central negative regulators of apoptosis is the Bcl-2 family of proteins (Adams et al., Science 281:1322 (1998); Reed, Adv. Pharmacol. 41:501 (1997); Reed et al., J. Cell. Biochem. 60:23 (1996)). Bcl-2 is the founding member of the family and was first isolated as the product of an oncogene. The Bcl-2 family now includes both anti-apoptotic molecules such as Bcl-2 and Bcl-xL and pro-apoptotic molecules such as Bax, Bak, Bid, and Bad. Bcl-2 and Bcl-xL are overexpressed in many types of human cancer (e.g., breast, prostate, colorectal, lung), including Non-Hodgkin's lymphoma, which is caused by a chromosomal translocation (t14, 18) that leads to overexpression of Bcl-2. This suggests that many cancer cell types depend on the elevated levels of Bcl-2 and/or Bcl-xL to survive the other cellular derangements that simultaneously both define them as cancerous or pre-cancerous cells and cause them to attempt to execute the apoptosis pathway. Also, increased expression of Bcl-2 family proteins has been recognized as a basis for the development of resistance to cancer therapeutic drugs and radiation that act in various ways to induce cell death in tumor cells.
Bcl-2 and Bcl-xL are thought to play a role in tumor cell migration and invasion, and therefore, metastasis. Amberger et al., Cancer Res. 58:149 (1998); Wick et al., FEBS Lett, 440:419 (1998); Mohanam et al., Cancer Res. 53:4143 (1993); Pedersen et al., Cancer Res., 53:5158 (1993). Bcl-2 family proteins appear to provide tumor cells with a mechanism for surviving in new and non-permissive environments (e.g., metastatic sites), and contribute to the organospecific pattern of clinical metastatic cancer spread. Rubio, Lab Invest. 81:725 (2001); Fernandez et al., Cell Death Differ. 7:350 (2000)). Anti-apoptotic proteins such as Bcl-2 and/or Bcl-xL are also thought to regulate cell-cell interactions, for example through regulation of cell surface integrins. Reed, Nature 387:773 (1997); Frisch et al., Curr. Opin. Cell Biol. 9:701 (1997); Del Bufalo et al., FASEB J. 11:947 (1997).
Therapeutic strategies for targeting Bcl-2 and Bcl-xL in cancer to restore cancer cell sensitivity and overcome resistance of cancer cells to apoptosis have been extensively reviewed (Adams et al., Science 281:1322 (1998); Reed, Adv. Pharmacol. 41:501 (1997); Reed et al., J. Cell. Biochem. 60:23 (1996)). Currently, Bcl-2 antisense therapy is in several Phase III clinical trials for the treatment of solid and non-solid tumors.
Gossypol is a naturally occurring double biphenolic compound derived from crude cotton seed oil (Gossypium sp.). Human trials of gossypol as a male contraceptive have demonstrated the safety of long term administration of these compounds (Wu, Drugs 38:333 (1989)). Gossypol has more recently been shown to have some anti-proliferative effects (Flack et al., J. Clin. Endocrinol. Metab. 76:1019 (1993); Bushunow et al., J. Neuro-Oncol. 43:79, (1999); Van Poznak et al., Breast Cancer Res. Treat. 66:239 (2001)). (−)-Gossypol and its derivatives recently have been shown to be potent inhibitors of Bcl-2 and Bcl-xL and to have strong anti-cancer activity (U.S. Patent Application Nos. 2003/0008924; 2004/0214902).
Several methods have been used to separate the (−)- and (+)-enantiomers of gossypol from enantiomeric mixtures. Derivatization of gossypol with optically active amines (e.g., S-1-methylphenethylamine, L-phenylalaminol, (+)-phenylalanine methyl ester) followed by separation of the diastereomers and hydrolysis of the derivatives has been reported. Yikang et al., Scientia Sinica 30:297 (1987); Sampath et al., J. Chem. Soc., Chem. Commun. 9:649 (1986); Matlin et al., Contraception 37:229 (1988). Chinese Patent No. 1017705B discloses the derivatization of gossypol using optically active primary amines, followed by separation of the enantiomers by chromatography or crystallization.
Gossypol is capable of forming a composition, e.g., a co-crystal or solvate, with many different solvent molecules in varying ratios. Typical compositions are those comprising gossypol with acetone or acetic acid. Compositions comprising gossypol and acetic acid are known in the art and commercially available (e.g., Sigma-Aldrich Corp., St. Louis, Mo.). Previous attempts to crystallize (−)-gossypol have resulted in crystals that are too poor for X-ray analysis (Gdaniec et al., “Gossypol,” in Comprehensive Supramolecular Chemistry (Atwood et al. eds.), Vol. 6, Pergamon) or in co-crystals of (−)-gossypol and acetone when using a solution of gossypol acetic acid in acetone (Dowd et al., J. Am. Oil Chem. Soc. 76:1343 (1999)) or in co-crystals of (−)-gossypol and 2,4-pentanedione (Dowd et al., J. Chem. Crystallogr. 34:559 (2004)).